Properties of Deiters' neurons and inhibitory synaptic transmission in the mouse lateral vestibular nucleus.

Deiters' neurons, located exclusively in the lateral vestibular nucleus (LVN), are involved in vestibulospinal reflexes, innervate extensor motoneurons that drive antigravity muscles, and receive inhibitory inputs from the cerebellum. We investigated intrinsic membrane properties, short-term plasticity, and inhibitory synaptic inputs of mouse Deiters' and non-Deiters' neurons within the LVN. Deiters' neurons are distinguished from non-Deiters' neurons by their very low input resistance (105.8 vs. 521.8 MΩ, respectively), long axons that project as far as the ipsilateral lumbar spinal cord, and expression of the cytostructural protein nonphosphorylated neurofilament protein (NPNFP). Whole cell patch-clamp recordings in brain stem slices show that most Deiters' and non-Deiters' neurons were tonically active (>92%). Short-term plasticity was studied by examining discharge rate modulation following release from hyperpolarization [postinhibitory rebound firing (PRF)] and depolarization [firing rate adaptation (FRA)]. PRF and FRA gain were similar in Deiters' and non-Deiters' neurons (PRF 24.9 vs. 20.2 Hz and FRA gain 231.5 vs. 287.8 spikes/s/nA, respectively). Inhibitory synaptic input to both populations showed that GABAergic rather than glycinergic inhibition dominated. However, GABAA miniature inhibitory postsynaptic current (mIPSC) frequency was much higher in Deiters' neurons compared with non-Deiters' neurons (∼15.9 vs. 1.4 Hz, respectively). Our data suggest that Deiters' neurons can be reliably identified by their intrinsic membrane and synaptic properties. They are tonically active and glutamatergic, have low sensitivity or "gain," exhibit little adaptation, and receive strong GABAergic input. Deiters' neurons also have minimal short-term plasticity, and together these features suggest they are well suited to a role in encoding tonic signals for the vestibulospinal reflex.NEW & NOTEWORTHY Deiters' neurons within the lateral vestibular nucleus project the length of the spinal cord and activate antigravity extensor muscles. Deiters' neurons were characterized anatomically and physiologically in mice. Deiters' neurons are tonically active, have homogeneous intrinsic membrane properties, including low input resistance, and receive significant GABAAergic synaptic inputs. Deiters' neurons show little modulation in response to current injection. These features are consistent with Deiters' neurons responding to perturbations to maintain posture and balance.

Reviewing the case for compromised spinal inhibition in neuropathic pain.

A striking and debilitating property of the nervous system is that damage to this tissue can cause chronic intractable pain, which persists long after resolution of the initial insult. This neuropathic form of pain can arise from trauma to peripheral nerves, the spinal cord, or brain. It can also result from neuropathies associated with disease states such as diabetes, human immunodeficiency virus/AIDS, herpes, multiple sclerosis, cancer, and chemotherapy. Regardless of the origin, treatments for neuropathic pain remain inadequate. This continues to drive research into the underlying mechanisms. While the literature shows that dysfunction in numerous loci throughout the CNS can contribute to chronic pain, the spinal cord and in particular inhibitory signalling in this region have remained major research areas. This review focuses on local spinal inhibition provided by dorsal horn interneurons, and how such inhibition is disrupted during the development and maintenance of neuropathic pain.

Aging alters signaling properties in the mouse spinal dorsal horn.

A well-recognized relationship exists between aging and increased susceptibility to chronic pain conditions, underpinning the view that pain signaling pathways differ in aged individuals. Yet despite the higher prevalence of altered pain states among the elderly, the majority of preclinical work studying mechanisms of aberrant sensory processing are conducted in juvenile or young adult animals. This mismatch is especially true for electrophysiological studies where patch clamp recordings from aged tissue are generally viewed as particularly challenging. In this study, we have undertaken an electrophysiological characterization of spinal dorsal horn neurons in young adult (3-4 months) and aged (28-32 months) mice. We show that patch clamp data can be routinely acquired in spinal cord slices prepared from aged animals and that the excitability properties of aged dorsal horn neurons differ from recordings in tissue prepared from young animals. Specifically, aged dorsal horn neurons more readily exhibit repetitive action potential discharge, indicative of a more excitable phenotype. This observation was accompanied by a decrease in the amplitude and charge of spontaneous excitatory synaptic input to dorsal horn neurons and an increase in the contribution of GABAergic signaling to spontaneous inhibitory synaptic input in aged recordings. While the functional significance of these altered circuit properties remains to be determined, future work should seek to assess whether such features may render the aged dorsal horn more susceptible to aberrant injury or disease-induced signaling and contribute to increased pain in the elderly.

Is more always better? How different 'doses' of exercise after incomplete spinal cord injury affects the membrane properties of deep dorsal horn interneurons.

Interneurons in the deep dorsal horn (DDH) of the spinal cord process somatosensory input, and form an important link between upper and lower motoneurons to subsequently shape motor output. Exercise training after SCI is known to improve functional motor recovery, but little is known about the mechanisms within spinal cord neurons that underlie these improvements. Here we investigate how the properties of DDH interneurons are affected by spinal cord injury (SCI) alone, and SCI in combination with different 'doses' of treadmill exercise training (3, 6, and 9wks). In an adult mouse hemisection model of SCI we used whole-cell patch-clamp electrophysiology to record intrinsic, AP firing and gain modulation properties from DDH interneurons in a horizontal spinal cord slice preparation. We find that neurons within two segments of the injury, both ipsi- and contralateral to the hemisection, are similarly affected by SCI and SCI plus exercise. The passive intrinsic membrane properties input resistance (Rin) and rheobase are sensitive to the effects of recovery time and exercise training after SCI thus altering DDH interneuron excitability. Conversely, select active membrane properties are largely unaffected by either SCI or exercise training. SCI itself causes a mismatch in the expression of voltage-gated subthreshold currents and AP discharge firing type. Over time after SCI, and especially with exercise training (9wks), this mismatched expression is exacerbated. Lastly, amplification properties (i.e. gain of frequency-current relationship) of DDH interneurons are altered by SCI alone and recover spontaneously with no clear effect of exercise training. These results suggest a larger 'dose' of exercise training (9wks) has a strong and selective effect on specific membrane properties, and on the output of interneurons in the vicinity of a SCI. These electrophysiological data provide new insights into the plasticity of DDH interneurons and the mechanisms by which exercise therapy after SCI can improve recovery.

Heteromeric α/ß glycine receptors regulate excitability in parvalbumin-expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.

KEY POINTS: Spinal parvalbumin-expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn. This study assessed the inhibitory regulation of the parvalbumin-expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability. Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin-expressing interneuron excitability. Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/ß subunit-containing receptors underlie both synaptic and tonic glycinergic currents. Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin-expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. ABSTRACT: The dorsal horn (DH) of the spinal cord is an important site for modality-specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin-expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch-clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine-mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/ß glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.

Distinct forms of synaptic inhibition and neuromodulation regulate calretinin-positive neuron excitability in the spinal cord dorsal horn.

The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH.

Intrinsic excitability differs between murine hypoglossal and spinal motoneurons.

Motoneurons differ in the behaviors they control and their vulnerability to disease and aging. For example, brain stem motoneurons such as hypoglossal motoneurons (HMs) are involved in licking, suckling, swallowing, respiration, and vocalization. In contrast, spinal motoneurons (SMs) innervating the limbs are involved in postural and locomotor tasks requiring higher loads and lower movement velocities. Surprisingly, the properties of these two motoneuron pools have not been directly compared, even though studies on HMs predominate in the literature compared with SMs, especially for adult animals. Here we used whole cell patch-clamp recording to compare the electrophysiological properties of HMs and SMs in age-matched neonatal mice (P7-P10). Passive membrane properties were remarkably similar in HMs and SMs, and afterhyperpolarization properties did not differ markedly between the two populations. HMs had narrower action potentials (APs) and a faster upstroke on their APs compared with SMs. Furthermore, HMs discharged APs at higher frequencies in response to both step and ramp current injection than SMs. Therefore, while HMs and SMs have similar passive properties, they differ in their response to similar levels of depolarizing current. This suggests that each population possesses differing suites of ion channels that allow them to discharge at rates matched to the different mechanical properties of the muscle fibers that drive their distinct motor functions.

In vivo characterization of colorectal and cutaneous inputs to lumbosacral dorsal horn neurons in the mouse spinal cord.

Chronic abdominal pain is a common symptom of inflammatory bowel disease and often persists in the absence of gut inflammation. Although the mechanisms responsible for ongoing pain are unknown, clinical and preclinical evidence suggests lumbosacral spinal cord dorsal horn neurons contribute to these symptoms. At present, we know little about the intrinsic and synaptic properties of this population of neurons in either normal or inflammed conditions. Therefore, we developed an in vivo preparation to make patch-clamp recordings from superficial dorsal horn (SDH) neurons receiving colonic inputs in naïve male mice. Recordings were made in the lumbosacral spinal cord (L6-S1) under isoflurane anesthesia. Noxious colorectal distension (CRD) was used to determine whether SDH neurons received inputs from mechanical stimulation/distension of the colon. Responses to hind paw/tail cutaneous stimulation and intrinsic and synaptic properties were also assessed, as well as action potential discharge properties. Approximately 11% of lumbosacral SDH neurons in the cohort of neurons sampled responded to CRD and a majority of these responses were subthreshold. Most CRD-responsive neurons (80%) also responded to cutaneous stimuli, compared with <50% of CRD-non-responsive neurons. Furthermore, CRD-responsive neurons had more hyperpolarized resting membrane potentials, larger rheobase currents, and reduced levels of excitatory drive, compared to CRD-non-responsive neurons. Our results demonstrate that CRD-responsive neurons can be distinguished from CRD-non-responsive neurons by several differences in their membrane properties and excitatory synaptic inputs. We also demonstrate that SDH neurons with colonic inputs show predominately subthreshold responses to CRD and exhibit a high degree of viscerosomatic convergence.

Electrical maturation of spinal neurons in the human fetus: comparison of ventral and dorsal horn.

The spinal cord is critical for modifying and relaying sensory information to, and motor commands from, higher centers in the central nervous system to initiate and maintain contextually relevant locomotor responses. Our understanding of how spinal sensorimotor circuits are established during in utero development is based largely on studies in rodents. In contrast, there is little functional data on the development of sensory and motor systems in humans. Here, we use patch-clamp electrophysiology to examine the development of neuronal excitability in human fetal spinal cords (10-18 wk gestation; WG). Transverse spinal cord slices (300 µm thick) were prepared, and recordings were made, from visualized neurons in either the ventral (VH) or dorsal horn (DH) at 32°C. Action potentials (APs) could be elicited in VH neurons throughout the period examined, but only after 16 WG in DH neurons. At this age, VH neurons discharged multiple APs, whereas most DH neurons discharged single APs. In addition, at 16-18 WG, VH neurons also displayed larger AP and after-hyperpolarization amplitudes than DH neurons. Between 10 and 18 WG, the intrinsic properties of VH neurons changed markedly, with input resistance decreasing and AP and after-hyperpolarization amplitudes increasing. These findings are consistent with the hypothesis that VH motor circuitry matures more rapidly than the DH circuits that are involved in processing tactile and nociceptive information.

Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing.

KEY POINTS: The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs. Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin-expressing neurons. Most calretinin cells (85%) exhibited large A-type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization-activated cation currents and low threshold T-type calcium currents, and tonic- or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord. Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin-expressing neurons suggest that they play distinct roles in spinal sensory processing circuits. ABSTRACT: Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

Electrophysiological characterization of spontaneous recovery in deep dorsal horn interneurons after incomplete spinal cord injury.

In the weeks and months following an incomplete spinal cord injury (SCI) significant spontaneous recovery of function occurs in the absence of any applied therapeutic intervention. The anatomical correlates of this spontaneous plasticity are well characterized, however, the functional changes that occur in spinal cord interneurons after injury are poorly understood. Here we use a T10 hemisection model of SCI in adult mice (9-10 wks old) combined with whole-cell patch clamp electrophysiology and a horizontal spinal cord slice preparation to examine changes in intrinsic membrane and synaptic properties of deep dorsal horn (DDH) interneurons. We made these measurements during short-term (4 wks) and long-term (10 wks) spontaneous recovery after SCI. Several important intrinsic membrane properties are altered in the short-term, but recover to values resembling those of uninjured controls in the longer term. AP discharge patterns are reorganized at both short-term and long-term recovery time points. This is matched by reorganization in the expression of voltage-activated potassium and calcium subthreshold-currents that shape AP discharge. Excitatory synaptic inputs onto DDH interneurons are significantly restructured in long-term SCI mice. Plots of sEPSC peak amplitude vs. rise times suggest considerable dendritic expansion or synaptic reorganization occurs especially during long-term recovery from SCI. Connectivity between descending dorsal column pathways and DDH interneurons is reduced in the short-term, but amplified in long-term recovery. Our results suggest considerable plasticity in both intrinsic and synaptic mechanisms occurs spontaneously in DDH interneurons following SCI and takes a minimum of 10 wks after the initial injury to stabilize.

Functional changes in deep dorsal horn interneurons following spinal cord injury are enhanced with different durations of exercise training.

KEY POINTS: Exercise training after spinal cord injury (SCI) enhances collateral sprouting from axons near the injury and is thought to promote intraspinal circuit reorganisation that effectively bridges the SCI. The effects of exercise training, and its duration, on interneurons in these de novo intraspinal circuits are poorly understood. In an adult mouse hemisection model of SCI, we used whole-cell patch-clamp electrophysiology to examine changes in the intrinsic and synaptic properties of deep dorsal horn interneurons in the vicinity of a SCI in response to the injury, and after 3 and 6 weeks of treadmill exercise training. SCI alone exerted powerful effects on the intrinsic and synaptic properties of interneurons near the lesion. Importantly, synaptic activity, both local and descending, was preferentially enhanced by exercise training, suggesting that exercise promotes synaptic plasticity in spinal cord interneurons that are ideally placed to form new intraspinal circuits after SCI. Following incomplete spinal cord injury (SCI), collaterals sprout from intact and injured axons in the vicinity of the lesion. These sprouts are thought to form new synaptic contacts that effectively bypass the lesion epicentre and contribute to improved functional recovery. Such anatomical changes are known to be enhanced by exercise training; however, the mechanisms underlying exercise-mediated plasticity are poorly understood. Specifically, we do not know how SCI alone or SCI combined with exercise alters the intrinsic and synaptic properties of interneurons in the vicinity of a SCI. Here we use a hemisection model of incomplete SCI in adult mice and whole-cell patch-clamp recording in a horizontal spinal cord slice preparation to examine the functional properties of deep dorsal horn (DDH) interneurons located in the vicinity of a SCI following 3 or 6 weeks of treadmill exercise training. We examined the functional properties of local and descending excitatory synaptic connections by recording spontaneous excitatory postsynaptic currents (sEPSCs) and responses to dorsal column stimulation, respectively. We find that SCI in untrained animals exerts powerful effects on intrinsic, and especially, synaptic properties of DDH interneurons. Plasticity in intrinsic properties was most prominent at 3 weeks post SCI, whereas synaptic plasticity was greatest at 6 weeks post injury. Exercise training did not markedly affect intrinsic membrane properties; however, local and descending excitatory synaptic drive were enhanced by 3 and 6 weeks of training. These results suggest exercise promotes synaptic plasticity in spinal cord interneurons that are ideally placed to form new intraspinal circuits after SCI.

Intrinsic and synaptic homeostatic plasticity in motoneurons from mice with glycine receptor mutations.

Inhibitory synaptic inputs to hypoglossal motoneurons (HMs) are important for modulating excitability in brainstem circuits. Here we ask whether reduced inhibition, as occurs in three murine mutants with distinct naturally occurring mutations in the glycine receptor (GlyR), leads to intrinsic and/or synaptic homeostatic plasticity. Whole cell recordings were obtained from HMs in transverse brainstem slices from wild-type (wt), spasmodic (spd), spastic (spa), and oscillator (ot) mice (C57Bl/6, approximately postnatal day 21). Passive and action potential (AP) properties in spd and ot HMs were similar to wt. In contrast, spa HMs had lower input resistances, more depolarized resting membrane potentials, higher rheobase currents, smaller AP amplitudes, and slower afterhyperpolarization current decay times. The excitability of HMs, assessed by "gain" in injected current/firing-frequency plots, was similar in all strains whereas the incidence of rebound spiking was increased in spd. The difference between recruitment and derecruitment current (i.e., ΔI) for AP discharge during ramp current injection was more negative in spa and ot. GABAA miniature inhibitory postsynaptic current (mIPSC) amplitude was increased in spa and ot but not spd, suggesting diminished glycinergic drive leads to compensatory adjustments in the other major fast inhibitory synaptic transmitter system in these mutants. Overall, our data suggest long-term reduction in glycinergic drive to HMs results in changes in intrinsic and synaptic properties that are consistent with homeostatic plasticity in spa and ot but not in spd. We propose such plasticity is an attempt to stabilize HM output, which succeeds in spa but fails in ot.

HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus.

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1-4), which confer distinct biophysical properties on the channel. Despite understanding the structure-function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several neurological diseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions.

Morphological, neurochemical and electrophysiological features of parvalbumin-expressing cells: a likely source of axo-axonic inputs in the mouse spinal dorsal horn.

Perception of normal bodily sensations relies on the precise regulation of sensory information entering the dorsal horn of the spinal cord. Inhibitory, axoaxonic, synapses provide a mechanism for this regulation, but the source of these important inhibitory connections remains to be elucidated. This study shows that a subpopulation of spinal interneurons that expresses parvalbumin and have specific morphological, connectivity and functional characteristics are a likely source of the inhibitory inputs that selectivity regulate non-noxious tactile input in the spinal cord. Our findings suggest that a loss of normal function in parvalbumin positive dorsal horn neurons may result in the development of tactile allodynia, where non-painful stimuli gain the capacity to evoke the sensation of pain.

Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord.

Neurons in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord process nociceptive information from skin, muscle, joints and viscera. Most of what we know about the intrinsic properties of SDH neurons comes from studies in lumbar segments of the cord even though clinical evidence suggests nociceptive signals from viscera and head and neck tissues are processed differently. This 'lumbar-centric' view of spinal pain processing mechanisms also applies to developing SDH neurons. Here we ask whether the intrinsic membrane properties of SDH neurons differ across spinal cord segments in both the developing and mature spinal cord. Whole cell recordings were made from SDH neurons in slices of upper cervical (C2-4), thoracic (T8-10) and lumbar (L3-5) segments in neonatal (P0-5) and adult (P24-45) mice. Neuronal input resistance (R(IN)), resting membrane potential, AP amplitude, half-width and AHP amplitude were similar across spinal cord regions in both neonates and adults (∼100 neurons for each region and age). In contrast, these intrinsic membrane properties differed dramatically between neonates and adults. Five types of AP discharge were observed during depolarizing current injection. In neonates, single spiking dominated (∼40%) and the proportions of each discharge category did not differ across spinal regions. In adults, initial bursting dominated in each spinal region, but was significantly more prevalent in rostral segments (49% of neurons in C2-4 vs. 29% in L3-5). During development the dominant AP discharge pattern changed from single spiking to initial bursting. The rapid A-type potassium current (I(Ar)) dominated in neonates and adults, but its prevalence decreased (∼80% vs. ∼50% of neurons) in all regions during development. I(Ar) steady state inactivation and activation also changed in upper cervical and lumbar regions during development. Together, our data show the intrinsic properties of SDH neurons are generally conserved in the three spinal cord regions examined in both neonate and adult mice. We propose the conserved intrinsic membrane properties of SDH neurons along the length of the spinal cord cannot explain the marked differences in pain experienced in the limbs, viscera, and head and neck.

The expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis?

The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin II type 1 and 2 receptors (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-ß1 (TGF-ß1), in early gestation (6-16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-ß1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT(1)R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.

Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse.

Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and ß subunits assembled in a 2:3 stoichiometry. The α1/ßheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24◦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⁻¹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 µM vs. 64 ± 11 µM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 µM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.

Pacemaker currents in mouse locus coeruleus neurons.

We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na(+) or oscillatory currents were detected. Ca(2+)-free and Ba(2+)-containing solutions failed to demonstrate a Ca(2+) current during the interspike interval, although a Ca(2+) current was activated at membrane potentials positive to -40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K(+) conductance during the interspike interval. Ca(2+)-activated K(+), inward rectifier and low-TEA (10 muM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca(2+) current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na(+) current and a high TEA-sensitive K(+) current. In contrast with rats, a persistent Ca(2+) current is not involved.

Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.

Spontaneous activity in medial vestibular nucleus (MVN) neurons is modulated by synaptic inputs. These inputs are crucial for maintaining gaze and posture and contribute to vestibular compensation after lesions of peripheral vestibular organs. We investigated how chronically attenuated glycinergic input affects excitability of MVN neurons. To this end we used three mouse strains (spastic, spasmodic, and oscillator), with well-characterized naturally occurring mutations in the inhibitory glycine receptor (GlyR). First, using whole-cell patch-clamp recordings, we demonstrated that the amplitude of the response to rapidly applied glycine was dramatically reduced by 25 to 90% in MVN neurons from mutant mice. We next determined how reduced GlyR function affected MVN neuron output. Neurons were classified using two schemas: (1) the shape of their action potential afterhyperpolarization (AHP); and (2) responses to hyperpolarizing current injection. In the first schema, neurons were classified as types A, B and C. The prevalence of type C neurons in the mutant strains was significantly increased. In the second schema, the proportion of neurons lacking post inhibitory rebound firing (PRF-deficient) was increased. In both schemas an increase in AHP amplitude was a common feature of the augmented neuron group (type C, PRF-deficient) in the mutant strains. We suggest increased AHP amplitude reduces overall excitability in the MVN and thus maintains network function in an environment of reduced glycinergic input.